title: Purification and stability of glutaryl-7-ACA acylase from Pseudomonas sp.
abstract: The enzyme glutaryl-7-ACA acylase from Pseudomonas sp. NCIMB 40474, produced by a recombinant Escherichia coli host, was purified to homogeneity. The enzyme is a tetramer composed of two couples of asymmetric dimers, each of them constituted of two subunits of mol wt 18 and 52 kDa, respectively. It was found that glutaric acid, one of the products of the substrate hydrolysis, is an effective acylase inhibitor. Between pH 6.0 and pH 10.0, the enzymatic activity is almost constant, but below pH 6.0 it progressively declines. The acylase activity decreased sharply as a function of guanidine HCl concentration. The loss is significant even at concentrations of denaturant lower than those causing unfolding, as suggested by UV spectroscopy and fluorescence emission studies. In these conditions (low denaturant concentration and low pH) the inactivation of the enzyme is caused by the tetramer dissociation into dimers. The lability of the quaternary structure of the enzyme is a key feature that must be taken into account for the improvement of the catalyst stability.
authors.affiliation: ENICHEM, Istituto Donegani, Italy.
authors.forename: E
authors.initials: E
authors.lastname: Battistel
authors.forename: D
authors.initials: D
authors.lastname: Bianchi
authors.forename: R
authors.initials: R
authors.lastname: Bortolo
authors.forename: L
authors.initials: L
authors.lastname: Bonoldi
mesh_terms: D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D019281:Dimerization; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D010399:Penicillin Amidase; D011487:Protein Conformation; D011549:Pseudomonas / Q000201:enzymology*; D013696:Temperature
chemical_list: D000581:Amidohydrolases; D010399:Penicillin Amidase; C046586:glutarylamidocephalosporanic acid acylase; D019791:Guanidine